This project will examine how calcineurin inhibitors, such as tacrolimus and cyclosporine, cause hypertension with potassium retention. In preliminary published studies, we showed that tacrolimus activates the thiazide-sensitive Na- Cl cotransporter (NCC) in the kidney. This effect was found to be essential for the resulting hypertension and potassium retention. Here we will examine the mechanisms involved. Calcineurin is a protein phosphatase and we will test whether it removes phosphates from NCC directly, to inhibit its actions, or whether the pathway involves calcineurin acting on intermediar proteins. We will determine whether other protein phosphatases, including protein phosphatases 1A, 2A, and 4 remove phosphates from NCC. Protein phosphatase inhibitor 1 (PPP1r1a) is a canonical target of calcineurin and is highly expressed along the distal nephron. In preliminary experiments by our collaborator, genetic deletion of this inhibitor was found to reduce the abundance of phosphorylated NCC in vivo. Therefore, we will test whether calcineurin regulates NCC activity via PPP1r1a. PPP1r1a is known to inhibit the actions of protein phosphatase 1A, which can act on the kinase SPAK. SPAK is the canonical NCC activating protein. Thus, we will test the over arching hypothesis that calcineurin inhibitors activate PPP1r1a in the distal nephron, leading to inhibition of PP1A, SPAK activation, and enhanced NCC activity. This model will be tested both in vitro, using a novel cell system that we developed recently, and in vivo, by knocking out key components of the signaling pathway. The proposal has substantial clinical implications, both in terms of patient treatment today, and in terms of drug development for tomorrow.